Marker for antidepressant therapy and methods related thereto

ABSTRACT

The present invention relates generally to methods for determining the effectiveness of ongoing antidepressant therapy via analysis of the association of G sα  with components of the plasma membrane or cytoskeleton of cells from peripheral tissues of the depressed individual as well as to methods involved in screening for effective antidepressant agents via their ability to cause a difference in the association of G sα  with components of the plasma membrane or cytoskeleton of cells.

Priority is claimed to U.S. Provisional Appl. No. 60/221,874, filed Jul.29, 2000, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods for determining theeffectiveness of antidepressant therapy in a depressed individual aswell as methods for detecting agents that possess antidepressantactivity.

2. Related Technology

Affective disorders are characterized by changes in mood as the primaryclinical manifestation. Major depression is one of the most commonmental illnesses and is often under diagnosed and frequentlyundertreated, or treated inappropriately. Major depression ischaracterized by feelings of intense sadness and despair, mental slowingand loss of concentration, pessimistic worry, agitation, andself-deprecation. Physical changes usually occur that include insomnia,anorexia and weight loss (or overeating) decreased energy and libido,and disruption of the normal circadian rhythms of activity, bodytemperature, and many endocrine functions. As many as 10-15% ofindividuals with this disorder display suicidal behavior during theirlifetime.

Antidepressant therapies are present in many diverse forms, includingtricyclic compounds, monoamine oxidase inhibitors, selective serotoninreuptake inhibitors (SSRIs), atypical antidepressants, andelectroconvulsive treatment. Antidepressant therapies vary widely inefficacy and the response of any given patient to a therapy isunpredictable. Unfortunately, therapy often proceeds for 1-2 monthsbefore it is established whether or not a specific modality of treatmentis effective. Thus, there remains a need for methods of ascertainingwhere the antidepressant therapy is effective in a depressed individualas well as a need for a method of screening for novel antidepressantagents.

SUMMARY OF THE INVENTION

The present invention is directed to methods for determining theeffectiveness of ongoing antidepressant therapy (during the early stagesof therapy) by whether there has been a modification of the associationof G_(sα) with components of the plasma membrane or cytoskeleton ofcells from peripheral tissues of the depressed individual.

Another aspect of the invention is directed to methods involved inscreening for effective antidepressant agents via their ability to alter(as compared to a control) the association of G_(sα) with components ofthe plasma membrane or cytoskeleton of cultured cells expressing Type VIadenylyl cyclase.

Other objectives and advantages of the invention may be apparent tothose skilled in the art from a review of the following detaileddescription, including any drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows detergent extraction of G_(sα) from C6-2B glioma membranestreated with antidepressants.

FIG. 2 shows co-localization of adenylyl cyclase activity with thepresence of G_(sα) in Triton X-100-extracted C6-2B cells fractionated ona sucrose density gradient.

FIG. 3 demonstrates that antidepressant treatment of C6-2B cells causesa shift in the localization of G_(sα) from a Triton X-100-insolublecaveolin-enriched domain to a more Triton X-100-soluble domain.

FIG. 4 shows fractional distribution of G_(sα) from C6-2B gliomamembranes treated with fluoxetine.

FIG. 5 demonstrates that chronic desipramine treatment of C6-2B gliomacells does not alter the overall shape of the cell. Cells were treatedand processed for microscopy as described. A representative image offive independent experiments is shown. Bar, 10 μm.

FIG. 6 shows that chronic desipramine treatment results in anenhancement of G_(sα) immunofluorescence in the cell body and a decreasein the cell processes and process tips. Untreated CD-2B glioma cells (Aand B) display ubiquitous staining of G_(sα) with an enhancement at theprocess tips (arrowheads) and cell processes (asterisks). Desipraminetreated cells C and D) show a decrease in G_(sα) staining at the processtips (arrowheads) and cell processes (asterisks) and simultaneouslydisplay an increase in cell body staining (arrows). Cells were treatedand prepared for microscopy as described previously. Bar=10 μm.

FIG. 7 shows an enlarged view of the process tips in control versusdesipramine treated C6-2B cells shown in FIG. 6. The process tips fromthe control cell in FIG. 6A were enlarged to show the intense G_(sα)staining (A and B) and the corresponding process tips of the desipraminetreated cell in FIG. 6C are shown to demonstrate the reduction of G_(sα)staining after antidepressant treatment© and D).

FIG. 8 demonstrates the qualitative differences between control anddesipramine-treated cells demonstrate a loss of G_(sα) staining in thecell processes and process tips.

FIG. 9 shows that G_(sα) does not undergo antidepressant inducedrelocalization. Untreated (A and B) and desipramine-treated© and D)cells show similar G_(sα) immunofluorescence profiles. There is stainingthroughout the cell body and processes of both sets of cells. The figureis typical of approximately 500 cells that were examined. Bar, 10 μm.

FIG. 10 shows that fluoxetine (10 μM) treatment for 3 days has effectssimilar to those of desipramine on G_(sα) cellular localization. Cellswere treated with fluoxetine (A) or chlorpromazine (B) and processed forconfocal microscopy as described. Like desipramine, fluoxetine treatmentresults in a drastic reduction of G_(sα) immunofluorescence in the cellprocess (arrow) and process tips (arrowhead), whereas chlorpromazinetreatment results in a uniform distribution of G_(sα) similar to control(compare to FIG. 6, A and B). The differential interference contrastimage to the right of the fluorescence image shows that these drugs haveno effect on global cell shape. Bar, 10 μm.

DETAILED DESCRIPTION OF THE INVENTION

Despite several decades of studies, the mechanism of antidepressantaction has not been clearly established. One of the most widely knownbiochemical effects of antidepressant treatment is an alteration in thedensity and/or sensitivity of several neurotransmitter receptor systems[Sulser, Adv. Biochem. Psychopharmacol., 39:249-261; 1984]). However,these effects do not fully explain the clinical efficacy of allantidepressants, mainly because of the dissociation between the timecourse of the change in the receptor numbers and their clinical timecourse [Rasenick et al., J. Clin. Psychiatry, 57:49-55; 1996].

Many studies searching for a common mechanism of antidepressant actionhave focused on postreceptor neuronal cell signaling processes aspotential targets of such action [Menkes et al. Science, 219:65-76,1983; Ozawa et al., Mol. Pharmacol., 36:803-808, 1989; Duman et al.,Arch. Gen. Psychiatry, 54:597-606, 1997; Takahashi et al., J. Neurosci.,19:610-616, 1999]. Much of this previous work has focused on thedownstream effects of antidepressant action, particularly thoseinvolving cAMP [Perez et al., Eur. J. Pharmacol., 172:305-316, 1989;Perez et al., Neuropsychopharmacology, 4:57-64, 1991; Duman et al.,Arch. Gen. Psychiatry, 54:597-606, 1997; Takahashi et al., J. Neurosci.,19:610-616, 1999]. Our focus is on the upstream events occurring at thepostsynaptic membrane involving G proteins and adenylyl cyclase.

Much of the current thinking about G protein-coupled receptors is basedon the idea of freely mobile receptors, G proteins, and effectors inwhich the specificity of their interaction is derived from thethree-dimensional structure of the sites of protein-proteininteractions. However, recent evidence indicates that an organizedinteraction of receptors, G proteins, and effectors with significantlimitations on lateral mobility [Kuo et al., Science, 260:232-234;1993]. Furthermore, these membrane proteins are associated with tubulinor other cytoskeletal proteins [Carlson et al., Mol. Pharmacol.,30:463-468, 1986; Rasenick et al., Adv. Second Messenger PhosphoproteinRes., 22:381-386, 1990; Wang et al., Biochemistry, 30:10957-10965,1991), which restrict distribution and mobility of G proteins to asurprising degree [Neubig, FASEB, 8:939-946; 1994]. These presence of awell organized network of cytoskeletal elements and the components ofneurotransmitter and hormonal G protein-mediated signal transductionsystems may play an important role in achieving this function.

Increasing evidence suggests that many species of heterotrimeric Gproteins are present in caveolin-enriched membrane domains, and caveolinhas been implicated as playing a major role in G protein-mediatedtransmembrane signaling [Okamoto et al., J. Biol. Chem., 273:5419-5422,1998]. Furthermore, Li et al. [J. Biol. Chem., 270:15693-15701, 1995]reported that the mutational or pharmacological activation of G_(sα)prevents its cofractionation with caveolin. Our data indicates thatantidepressant treatment of C6-2B cells causes a shift in thelocalization of G_(sα) from a caveolin-enriched domain to a more TritonX-100-soluble fraction (e.g., see FIG. 3). Such data are consistent withthe our finding that chronic antidepressant treatment alters theassociation between G_(sα) and some specific membrane component.

Multiple neural dysfunctions may exist in patients with depressivedisorders, and there are likely to exist multiple molecular targets forantidepressants. The ability of different classes (data disclosedherein) of antidepressants to show the same effect on the redistributionof G_(sα) in the plasma membrane indicates convergence.

In view of the foregoing discussion and by way of illustration of theinvention, the examples describe methods for determining theeffectiveness of ongoing antidepressant therapy by whether there hasbeen a modification of the association of G_(sα) with components of theplasma membrane or cytoskeleton of cells from peripheral tissues of thedepressed individual as well as methods methods involved in screeningfor effective antidepressant agents via their ability to alter (ascompared to a control) the association of G_(sα) with components of theplasma membrane or cytoskeleton of cultured cells expressing Type VIadenylyl cyclase.

The invention is illustrated by the following Examples, which are notintended to limit the scope of the invention as recited in the claims.

Example 1 provides methods and materials for experiments disclosed inExamples 2-8.

Example 2 describes results wherein the amount of G_(sα) in C6-2B gliomacell membranes is not altered by antidepressant treatment.

Example 3 provides results wherein detergent extraction of G_(sα) fromC6-2B glioma membrane is increased by antidepressant treatment.

Example 4 sets forth results wherein detergent extraction of G_(sα) fromC6-2B glioma membrane is increased by antidepressant treatment, withdisparate antidepressants.

Example 5 provides results showing that antidepressant treatmentincreases Triton X-100 solubility of G_(sα) from rat synaptic membrane.

Example 6 provides results with respect to sucrose density sedimentationof adenylyl cyclase activity in control and desipramine-treated C6-2Bcells

Example 7 describes results showing that antidepressant treatmentdecreases the colocalization of G_(sα) with triton-insoluble,caveolin-enriched membrane domains and antidepressant-enhanced mobilityis unique to G_(s).

Example 8 sets forth results of experiments showing thatantidepressant-enhanced mobility is unique to G_(sα).

Example 9 provides methods and materials for experiments disclosed inExamples 10-13.

Example 10 sets forth results from experiments showing that chronicantidepressant leads to a shift in cellular localization of G_(sα).

Example 11 provides results from experiments that show thatantidepressant-induced G protein α subunit cellular relocalization isspecific to G_(sα).

Example 12 sets forth results that show that (1) fluoxetine treatmentalso promotes G_(sα) migration and (2) chlorpromazine did not inducemigration.

Example 13 sets forth methods for screening for the effectiveness ofantidepressant therapy as well as screening for agents havingantidepressant activity.

EXAMPLE 1 Methods and Materials

Set forth below are methods and materials for experiments disclosed inExamples 2-8.

Cell/Tissue Preparation

C6-2B cells (between passages 20 and 40) were grown in 175-cm² flasks inDulbecco's modified Eagle medium, 4.5 g of glucose/L, 10% bovine serum,in a 10% CO₂ atmosphere, at 37° C. for 3 days after splitting. As 5 μMfor 5 days and 10 μM for 3 days of antidepressant treatment had asimilar effect on the Gpp(NH)_(p)— or forskolin-stimulated adenylylcyclase activity in the C6-2B cells [(hen et al., J. Neurochem.,64:724-732, 1995], the latter paradigm was used and cells were treatedwith 10 μM drug for 3 days. Media containing drugs (fluoxetine,amtriptyline, iprindole, desipramine, or chlorpromazine) were added todifferent flasks, and the media were changed daily. During the period ofexposure to antidepressants, no morphological change in the cells wasobserved. After treatment, the cells were incubated in drug-free mediafor 1 h before harvesting by scraping with a rubber policeman inHEPES-sucrose buffer [15 mM HEPES, 0.25 M sucrose, 0.3 mMphenylmethylsulfonyl fluoride (PMSF), 1 mM EGTA, and 1 mM dithiothreitol(DTT), pH 7.5], C6-2B membranes were prepared as described [Rasenick andKaplan, FEBS Lett. 207:296-301, 1986] and stored under liquid N₂ untiluse. Male Sprague-Dawley rats weighing 150-200 g were fed ad libitum andmaintained in a 12-h light/dark cycle. The method of antidepressanttreatment has been described previosuly [Ozawa and Rasenick, Mol. Pharm.36:803-838, 1989]. In brief, animals were treated with desipramine orfluoxetine (10 mg/kg i.p.) once daily for 21 days; the control groupreceived only saline injection daily for 21 days. Rat cerebral cortexmembranes were prepared according to the method of Rasenick et al.[Nature, 294:560-562; 1981].

Membrane Protein Extraction

Membrane proteins were extracted sequentially from C6-2B or rat synapticmembranes as described [Yan et al., J. Neurochem., 66:1489-1495; 1996].These membranes were stirred on ice in HEPES (15 mM, pH 7.4) containing1% Triton X-100 for 60 min followed by centrifugation at 100,000 g for60 min at 4° C. The supernatant was reserved (Triton X-100 extract), andthe resulting pellet was resuspended in Tris (15 mM, pH 7.4) containing1.4% Triton X-114 and 150 mM NaCl. The solution was stirred for 60 minat 4° C. and centrifuged for 30 min at 100,000 g in the cold. Thesupernatant was saved (Triton X-114 extract). These supernatants andremaining pellet (remainder) were subjected to sodium dodecyl sulfate(SDS)-polyacrylamide gel electophoresis (PAGE) using 10% gels. All ofthe extraction buffers contained 1 mM PMSF and 1 mM EGTA.

Cell Fractionation by Sucrose Density Gradient Sedimentation

C6-2B cells treated as described above were used to prepareTriton-insoluble, caveolin-enriched membrane fractions by the procedureof Li et al. [J. Biol. Chem., 270:15693-15701; 1995] with minormodifications. In brief, C6-2B cells were harvested into 0.75 ml ofHEPES buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.3 mM PMSF)containing 1% Triton X-100. Homogenization was carried out with 10strokes of a Potter-Elvehjem homogenizer. The homogenate was adjusted to40% sucrose by the addition of an equal volume of 80% sucrose preparedin HEPES buffer and placed at the bottom of an ultracentrifuge tube. Astep gradient containing 30, 15, and 5% sucrose was formed above thehomogenate and centrifuged at 50,000 rpm for 20 h in an SW65 rotor(240,000 g). Two or three opaque bands confined between the 15 and 30%sucrose layers were harvested, diluted threefold with HEPES buffer, andpelleted in a microcentrifuge at 16,000 g. The pellet was resuspended inHEPES buffer and identified as the Triton-insoluble fraction. The 40%sucrose region of the gradient was saved as the Triton-soluble fraction.In separate experiments, the same conditions were used, but instead ofisolating the three opaque bands, 100-111 fractions were collected andassayed for G_(s) content and adenylyl cyclase activity.

Assay of Adenylyl Cyclase Activity

Adenylyl cyclase was assayed as described previously [Rasenick et al.,Brain Res., 488:105-113; 1989]. Each sucrose gradient fraction wasassayed under both basal and stimulated (10 μM forskolin) conditions for10 min at 30° C. in 100 μl of medium containing 15 mM HEPES, pH 7.5,0.05 mM ATP, [α-³²P]ATP (5×10⁶ cpm/tube), 5 mM MgCl₂, 1 mM EGTA, 1 mMDTT, 0.5 mM cyclic AMP (cAMP), 60 mM NaCl, 0.25 mg/ml bovine serumalbumin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 U of adenosinedeaminase/ml, and a nucleotide triphosphate regenerating systemconsisting of 0.5 mg of creatine phosphate, 0.14 mg of creatinephosphokinase, and 15 U of myosin kinase/ml. The reaction was stopped byaddition of 0.1 ml of a solution containing 2% SDS, 1.4 mM cAMP, and 40mM ATP, and the [³²P]cAMP formed was isolated by the method of Salomon(1979) using [³H]cAMP to monitor recovery. All assays were performed intriplicate.

Immunoblotting

Rat cortex membrane, C6-2B cell membranes, or Triton extracts of eachmembrane preparation were subjected to SDS-PAGE followed byelectrotransfer to polyvinylidene difluoride (PVDF) membrane. The PVDFmembrane was incubated with a phosphate-buffered saline/Tween 20 Buffer(140 mM NaCl, 27 mM KCl, 81 mM Na₂HPO₄, 15 mM KH₂PO₄, 0.1% Tween 20, pH7.4) and 3% bovine serum albumin. The membrane was then incubated withpolyclonal rabbit antisera against the various G protein subunits [RM(G_(sα)) or 116 (G_(i1), G_(i2), G_(i3α))] at a 1:25,000 (RM) or 1:5,000(116) dilution (see below for source of antibodies). The PVDF membraneswere washed three times and incubated with a dilution of 1:5,000 of thesecond antibody [horseradish peroxidase-linked anti-rabbit IgG F(ab′)₂(Amersham)]. Immunoreactivity was detected with an enhancedchemiluminescence (ECL) western blot detection system (Amersham) inaccord with the manufacturer's instructions. The developedautoradiographs were analyzed by densitometry.

G Protein Purification and Quantification

G_(sα) (His) His₆ was purified, from Escherichia coli expressing therecombinant gene for that protein, by a modification of the method ofGilman [Lee et al., Methods Enzymol., 237:146-164; 1994]. In brief, thebacteria were grown overnight in an enriched medium (2% tryptone, 1%yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mM KH₂PO₄, pH 7.2)containing 50 μg/ml ampicillin 8 L (8×1 L in 2-L Erlenmeyer flasks).Bacteria were collected by centrifugation, cells were lysed bysonication, the cleared cell lysate was loaded onto a Ni-NTA resincolumn (QiaGen), and the protein was eluted with a step gradient of 20to 60 mM imidazole. G_(sα) protein was purified further byhigh-performance liquid chromatography with Resource Q chromatography(Pharmacia) and hydroxylapatite chromatography. Fractions containingG_(sα) protein were identified by labeling with[α-³²P]P³-(4-azidoanalido)-P¹-5′-GTP and immunoblotting. The G_(sα)was >98% pure as identified by silver stain of the gels.

Purified recombinant G_(sαL) (long form of G_(sα); 1-10 ng/lane) wassubjected to SDS-PAGE and transferred to PVDF membranes. The blots wereincubated with antibody against G_(sα) (RM) and processed by ECL. Filmswere analyzed on a Molecular Dynamics densitometer, and the volume ofeach band was quantified and used to make a standard curve.

Materials and Data Analysis

All detergents were obtained from Pierce, Anti-G protein antibodies werefrom Drs. David Manning (116; University of Pennsylvania) and Allen M.Spiegel (RM; National Institutes of Health). Caveolin antibody wasobtained from Transduction Laboratories (Lexington, Ky., U.S.A.) cloneno 2297. All other reagents used were of analytical grade.

Further, antibodies for use in the protocols disclosed herein may alsobe manufactured by well known methods [Antibodies, A Laboratory Manual,Harlow and Lane, Cold Spring Harbor Laboratory; ISBN: 0879693142].

Data were analyzed for statistical significance using Scheffé's test orBonferroni's multiple comparison test after a one-way ANOVA test ortwo-tailed t test. Values of p<0.05 were taken to indicate significance.

EXAMPLE 2 The Amount of G_(Sα) In C6-2B Glioma Membrane is not Alteredby Antidepressant Treatment

Experiments were conducted to determine whether G_(sα) is quantitativelyaltered by antidepressant therapy. For quantification of G_(sα) in C6-2Bmembrane, a standard curve was created using purified recombinantG_(sα). Pure G_(sα) (1-20 ng/well) was subjected to SDS-PAGE andtransferred to a PVDF membrane, G_(sα) was immunodetected by antiseraagainst the as subunit of G protein.

To estimate the effect of antidepressant treatment on the amount ofG_(sα) in C6-2B membranes, equal amounts (10 μg) of C6-2B gliomamembrane proteins in control and chronically (3 days)antidepressant-treated groups were subjected to SDS-PAGE. G_(sα) wasimmunodetected by antisera against the αs subunit of G protein. Table 1summarizes the quantity of G proteins in the membrane of C6-2B cellsafter exposure to 10 μM (3 days) amitriptyline, iprindole, or fluoxetine(values are expressed as means±standard error of the mean of 4-5experiments. As shown in Table 1, no significant differences weredetected (p>0.05) between antidepressant-treated and control groups.TABLE 1 Effect of antidepressant treatment on the content of G proteinin C6-2B glioma membranes Subunit of G protein amount (ng/10 μg ofmembrane protein) G protein Control Iprindole Amitriptyline FluoxetineαsL 8.35 ± 0.05 8.20 ± 0.20 8.13 ± 0.17 7.23 ± 0.56 αsS 5.15 ± 0.28 4.70± 0.12 4.98 ± 0.21 5.65 ± 0.74 Total 13.50 ± 0.27  12.93 ± 0.36  13.10 ±0.34  12.49 ± 0.67 

EXAMPLE 3 Detergent Extraction of G_(Sα) from C6-2B Glioma Membrane isIncreased by Antidepressant Treatment

In order to determine whether detergent extraction of G_(sα) from cellstreated with antidepressants increased the following experiment wasundertaken.

C6-2B cells were treated chronically with iprindole, amitriptyline, orchlorpromazine (10 μM, 3 days) and harvested. A membrane-enrichedfraction was prepared (see Example 1), and membrane proteins wereextracted sequentially with Triton X-100 (Tx 100) and Triton X-114 (Tx114). Equal amounts of these extracts were subjected to SDS-PAGE andtransferred to PVDF membrane. The long and short forms of G_(sα)(G_(sαL) and G_(sαS)) from different fractions were identified byimmunodetection. A representative immunoblot (FIG. 1) shows theredistribution of G_(sαL) and G_(sαS) in the plasma membrane afterchronic antidepressant treatment (i.e., the effect of the tricyclicantidepressant (amitriptyline) and an atypical, non-reuptake-inhibitingantidepressant (iprindole) on the redistribution). The data indicateG_(sα) was shifted to the less hydrophobic fraction (Triton X-100extract) from the more hydrophobic fraction (Triton X-114 extract)subsequent to treatment with antidepressant G_(sα) exists in four splicevariations that migrate as a long form (G_(sαL)) and short form(G_(sαS)) (the two long and two short variants are not resolved from oneanother). G_(sαL) in the Triton X-114 fraction was significantly lowerin the iprindole- and amitriptyline-treated groups than in the controlgroup. G_(sαS) also showed a tendency to migrate to the less hydrophobicdomain (Triton X-100 extract) from a more hydrophobic domain of theplasma membrane.

EXAMPLE 4 Disparate Antidepressants Have Similar Effects

Experiments were conducted according to the method set forth in Example1 to assess the effects of different antidepressants on the detergentextraction of G_(sα) from C6-2B glioma cells. Three differentantidepressants comprised of the tricyclic antidepressant(amitriptyline), the non-reuptake inhibitor (iprindole), and SSRI(fluoxetine) were used. Membranes were prepared from C6-2B glioma cellsthat had been exposed to a 10 μM concentration of the indicatedantidepressant drug for 3 days.

The ratios of the percentage of G_(sα) extracted by the two detergentsin the treatment versus control groups were compared and are set forthin Table 2 as percentage of control (i.e., each Triton X-100 extract wascompared with the untreated Triton X-100 extract; the same was done forTriton X-114 extracts as well as the remainder). The values shown aremeans±standard error of the mean of 4-5 experiments.

The different antidepressants achieved similar effects on G_(sα), i.e.,G_(sα) shifted to the less hydrophobic, Triton X-100 fraction subsequentto antidepressant treatment. In contrast, chlorpromazine, which is atricyclic compound but not an antidepressant, did not exert theseeffects. The total amount of G_(sα) was not changed by antidepressanttreatment, membrane preparation, or detergent extraction.

Other experiments showed that amphetamine, which is known to blockneurotransmitter uptake but does not have antidepressant activity, wasalso without effect. TABLE 2 Effects of antidepressants on detergentextraction of G_(sα) from C6-2B cells % of corresponding value incontrol group Extraction G_(sα) Antidepressant subunit Triton X-100Triton X-114 Remainder Iprindole G_(sαL) 128.0 ± 3.8^(a)  75.3 ± 2.2^(b) 87.7 ± 9.3 G_(sαS) 146.6 ± 13.3^(b)  74.7 ± 0.9^(b) 112.0 ± 1.9^(a)Amitriptyline G_(sαL) 120.3 ± 3.7^(b)  84.5 ± 0.8^(b)  83.4 ± 14.5G_(sαS) 140.3 ± 9.3^(b)  92.7 ± 3.8  82.6 ± 5.1^(a) Fluoxetine G_(sαL)146.4 ± 12.7^(a)  84.4 ± 2.9^(b)  90.8 ± 34.2 G_(sαS) 215.9 ± 30.9^(a) 92.2 ± 1.3^(b)  99.7 ± 3.1 Chlorpromazine G_(sαL) 107.4 ± 8.3 105.7 ±6.5  83.8 ± 22.0 G_(sαS) 113.4 ± 10.7 102.2 ± 15.6 100.4 ± 31.6^(a)p < 0.05,^(b)p < 0.01 by two-tailed t test

EXAMPLE 5 Antidepressant Treatment Increases Triton X-100 Solubility ofG_(Sα) from Rat Synaptic Membrane

Rats were treated with antidepressant via intraperitoneal injection oncedaily for 21 days, control animals received an equal number and volumeof injection of saline. Cerebral cortices from each group were removed.Rat synaptic membrane-enriched fractions were prepared (see Example 1).The ratios of the percentage of G_(sα) extracted by the two detergentsin the treatment versus control groups were compared and are set forthin Table 3 as percentage of control (i.e., each Triton X-100 extract wascompared with the untreated Triton X-100 extract, the same wascomparison was undertaken for Triton X-114 extracts as well as theremainder). The values shown are means±standard error of the mean of 4-6experiments.

Results indicate that both desipramine and fluoxetine treatment caused asignificant increase of G_(sαL) in the Triton X-100 extract and aconcomitant decrease in that protein in the Triton X-114 extract. Underno circumstances did the antidepressant treatment alter the amount ofG_(sα). The sum of G_(sα) immunoreactivity of the three fractions wasnot changed. Previous studies had demonstrated that a 1-day treatment ofcells or a 1-week treatment of rats was without effect in any of theparameters examined [Ozawa et al., J. Neurochem., 56:330-338, 1991; Chenet al., J. Neurochem., 64:724-732, 1995]). Similar short-term treatmentswith fluoxetine were also without effect. TABLE 3 Effects of chronicantidepressants on detergent extraction of G_(sα) from rat synapticmembrane % of corresponding value in control group ExtractionAntidepressant G_(sα) treatment Subunit Triton X-100 Triton X-114Remainder Desipramine G_(sαL) 128.2 ± 9.5^(a) 77.3 ± 8.8^(a) 66.0 ±10.2^(a) G_(sαS) 146.4 ± 9.6^(b) 71.7 ± 6.8^(a) 84.9 ± 25.7 FluoxetineG_(sαL) 118.1 ± 3.4^(a) 81.9 ± 4.5^(a) 53.8 ± 6.9^(a) G_(sαS) 153.8 ±39.9 91.6 ± 6.3 45.2 ± 8.4^(a)^(a)p < 0.05,^(b)p < 0.01 by two-tailed t test.

EXAMPLE 6 Sucrose Density Sedimentation of Adenylyl Cyclase Activity inControl and Desipramine-Treated C6-2B Cells

Membranes from control and 10 μM desipramine-treated C6-2B glioma cellswere solubilized in Triton X-100 and run on a discontinuous sucrosedensity gradient. Fractions were collected and assayed for adenylylcyclase activity and for the presence of G_(sα) by SDS-PAGE andimmunoblotting.

FIG. 2A shows adenylyl cyclase activity was measured on sucrose densitygradient fractions from control (circles) and desipramine-treated(squares) C6-2B cells under basal (open symbols) andforskolin-stimulated (filled symbols) conditions. FIG. 2B shows a G_(sα)immunoblot corresponding to the assayed fractions in FIG. 2A.

FIG. 2A demonstrates an increase in forskolin-stimulated adenylylcyclase activity for both control and desipramine-treated cells (notethe corresponding increase in G_(sα) and adenylyl cyclase activity infractions 10 and 12 of the desipramine-treated group). Further, there isalmost a twofold increase in enzyme activity in the desipramine-treatedcells compared with control in fractions 11 and 12. Basal adenylylcyclase activity is unchanged in either group. The increase inforskolin-stimulated adenylyl cyclase activity corresponds to anincrease in G_(sα) in these same fractions (FIG. 2B, fraction 12).

EXAMPLE 7 Antidepressant Treatment Decreases the Colocalization ofG_(Sα) with Triton-Insoluble, Caveolin-Enriched Membrane Domains andAntidepressant-Enhanced Mobility is Unique to G_(s)

Recently, it has been reported that caveolin, a Triton-insolublemembrane protein, participates in plasma membrane coupling events(Okamoto et al., J. Biol. Chem., 273:5419-5422; 1998). To evaluate theinteraction between G_(sα) and caveolin-enriched membrane domains, C6-2Bcell lysates were subjected to sucrose density gradient fractionationand divided into a low-density, Triton X-100-insoluble fraction, whichis dramatically enriched in caveolin, and a Triton X-100-solublefraction, which contains the majority of other membrane proteins.Results are set forth in FIG. 3.

FIG. 3A reveals a set of representative immunoblots of the distributionof G_(sα) after sucrose density gradient fractionation. As shown,chronic antidepressant treatment of C6-2B cells causes a shift in thelocalization of G_(sα) from a caveolin-enriched domain to a more TritonX-100-soluble domain. The amount of G_(sα) in the soluble fraction ofdesipramine- and fluoxetine-treated cells was consistently three to fivetimes greater than the amount in the caveolin-enriched fraction, makinga direct measurement of the shift from one domain to the other nearlyimpossible. FIG. 3C shows the percent change in G_(sα) in thecaveolin-enriched Triton-insoluble fraction normalized to equalcaveolin. FIG. 3B shows a typical caveolin immunoblot used for thisnormalization. There was only a 5-10% difference in the amount ofcaveolin isolated in the Triton-insoluble domain between theexperimental groups, and very little caveolin was found in the solublefraction. Both desipramine and fluoxetine treatment decreased the amountof G_(sα) in the caveolin-enriched fraction by ˜50%. In contrast,chlorpromazine caused a reduction of no more than 25% of G_(sα) in thecaveolin-enriched fraction. Results were determined to be significant byone-way ANOVA (***p<0.0001), as well as Bonferroni's (***p<0.001)multiple comparison tests.

EXAMPLE 8 Antidepressant-Enhanced Mobility is Unique of G_(Sα)

Experiments were conducted (as described in Example 1) to ascertain theeffect of antidepressant therapy on the mobility of G_(iα) from C6-2Bglioma cells. Specifically, C6-2B cells were treated with fluoxetine (10μM, 3 days) and harvested. A membrane-enriched fraction was prepared,and membrane protein was extracted sequentially with Triton X-100 (Tx100) and Triton X-114 (Tx 114). Equal amounts of these extracts weresubjected to SDS-PAGE and transferred to PVDF membrane. The PVDFmembrane was incubated with polyclonal rabbit antisera against G_(iα1),and G_(iα2a). Immunoreactivity was detected with an ECL western blotdetection system.

Results, which are set forth in FIG. 4, indicated that unlike G_(sα),G_(iα) did not migrate to a less hydrophobic membrane fraction afterantidepressant treatment.

EXAMPLE 10 Methods and Materials

Set forth below are method and materials used in experiments discussedin Examples 10-13.

Cell Culture

C6-2B cells (between passages 30 and 50) were plated onto coverslips andallowed to attach overnight in Dulbecco's modified Eagle's medium, 4.5g/l glucose, 10% bovine serum, and 100 μg/ml penicillin and streptomycinat 37° C. in a humidified 10% CO₂ atmosphere. As reported previously,desipramine treatment regimens of 3 μM for 5 days and 10 μM for 3 daysyielded similar biochemical results (Chen and Rasenick, 1995b).Therefore, the latter treatment paradigm was used in these experimentsbecause it was easier to maintain the cell cultures for 3 days. In someinstances, 10 μM fluoxetine was used. The culture media and drug werechanged daily. Neither desipramine nor fluoxetine treatment altered cellgrowth (as determined by the confluence of the cell monolayer and totalprotein estimation) or cell viability (as determined by4,6-diamidino-2-phenylindole staining and visualization under afluorescence microscope with UV light). During the treatment duration,no morphological changes were observed in the cells. After the treatmentduration, the cells were incubated in drug-free media for 45 to 60minutes before fixation.

Indirect Immunofluorescence Laser Scanning Confocal Micros Copy

After treatment, cells were washed once with phosphate-buffered saline(PBS, 136 nM NaCl, 2.6 mM KCl, 5.4 mM Na_(a)PO₄ 7H₂O, pH 7.4) and fixedwith ice-cold methanol for 10 minutes. Cells were then wahed three timeswith PBS followed by 2 h of blocking in 5% normal goat serum/0.2% fishskin gelatin in PBS. Primary antibody was added for 1.5 h, G_(sα)/RM1(PerkinElmer Life Sciences, Boston, Mass.) 1:50 and Goα (Santa CruzBiotechnology, Santa Cruz, Calif.) 2 μg/ml, followed by three washeswith PBS. Oregon Green-labeled secondary antibody (Molecular Probes,Eugene, Oreg.) was added at a concentration of 8 μg/ml for 1 h followedby three PBS washes. The coverslips were mounted onto slides withVectashield (Vector Laboratories, Burlingame, Calif.) containingdiamidino-2-phenylindole as a mounting medium. Images were acquiredusing a Zeiss LSM510 laser-scanning confocal microscope (Carl ZeissInc., Thornwood, N.Y.). A single 488-nm beam from an argon/krypton laserwas used for excitation of the Oregon Green. Differential interferencecontrast images were also acquired. Five experiments were performed andcoverslips were examined. Approximately 2100 cells

from control and desipramine-treated coverslips were counted by twoinvestigators blind to the experimental conditions over the course ofthe five experiments.

Fluorescence Quantification

The cellular distribution of G_(sα) was quantified in confocal imagedC6-2B cells using NIH-Image software (http://rsbinfonihpov/nih-image) asdescribe previously [Southwell et al., Cell Tissue Res., 292:37-45,1998; Jenkinson et al., Br. J. Pharmacol., 126:131-136, 1999]. Images of9×1 μm optical, planar sections taken from four randomly selectedcontrol and four randomly selected desipramine-treated cells werecaptured and the middle five sections from each cell were quantified.Total cellular G_(sα) fluorescence was measured by counting the numberof pixels with intensity above threshold (determined by minimumintensity above background in this case 50 pixels). The areas ofintensity were numbered and divided visually into those localized to thecell body and those localized to the processes and process tips. Thetotal from each region was divided by the total cell pixel intensity andexpressed as a percentage of the total. This was done for each sectionof each cell and the sections were averaged per cell to give an averagepercentage total per cell.

In a separate investigation, seven sets of 300 cells each from controlgroup and desipramine-treated cells from five experiments were countedto determine the primary localization (processes and process tips orcell body) of G_(s)α within these cells. The majority of the cellsstained positively for G_(sα) throughout the entire cell, but there wasusually an enhancement in one of these regions. Overly flattened andfragmented cells were omitted from counting, as were cells that did notdisplay processes. The counts are expressed as the ratio of process andprocess tip localization/cell body localization.

Data Analysis

Images were evaluated by two investigators blinded to the treatmentcondition. Student's t test was performed for statistical analysis.Values of p<0.05 were taken to indicate significance.

EXAMPLE 10 Chronic Antidepressant Treatment Leads to a Shift in theCellular Localization of G_(sα)

Studies were conducted to determine whether a shift in cellularlocalization of G_(sα) occurred in C6-2B cells subjected toantidepressants. C6-2B glioma cells were treated with the tricyclicantidepressant desipramine (10 μM) for three days and were then examinedby laser scanning confocal microscopy to visualize these changes inmembrane localization (See Example 9 for specific experimentalprotocol).

Examination of 300 to 500 control and desipramine-treated cells by threeindependent researchers revealed that desipramine treatment did notalter the overall structure of C6-2B cells (FIG. 5), but drasticallyreduced the presence of G_(sα) in the process tips (FIG. 6, arrowheadsand FIG. 7). In addition, there was an increase in the presence ofG_(sα) within the cell body of many of the desipramine-treated cells(FIG. 6, arrows), as well as a decrease within the cell processesthemselves (FIG. 6, asterisks). In some instances, there was an intenseclustering of G_(sα) staining in the cell body (FIG. 6C, arrows), butthe majority of the cells did not exhibit such a focal increase inG_(sα) staining.

Twenty-one hundred cells from each group (control versusdesipramine-treated) over a series of five experiments were examined toquantify the extent of the antidepressant effect. The cells were groupedinto two categories: those that displayed intense staining at theprocess tips as well as overall staining in the processes and cell body(category A) versus those that displayed intense staining in the cellbody region and decreased process and process tip staining (category B).Abnormal cells or those not displaying processes were not included inthe cell count. Cells (300-450) were counted per experiment and theratio of category A cells to category B cells for each group is shown inFIG. 8.

Twice as many control cells (64%) displayed G_(sα) staining at theprocess tips and throughout the entire cell than those treated withdesipramine (32%). This demonstrates that G_(sα) relocalization is notan all-or-none response to antidepressant treatment and that some cellsmay be more responsive to treatment than others.

To determine quantitative differences between the groups, five 1 μmoptical, planar sections through each of four cells in each group wereexamined by confocal microscopy and the digital images were captured.These images were then analyzed using the program NIH Image [Southwellet al., Cell Tissue Res., 292:37-45, 1998; Jenkinson et al., Br. J.Pharmacol., 126:131-136, 1999]. These experiments were undertaken toaccount for changes in G_(sα) localization at different focal planes ofthe cell. The percentage of G_(sα) localized to the cellular processesand process tips of control versus treated cells were compared bydividing the pixel density above threshold in these regions by the totalcellular pixel density (Table 4). There was a three-fold decrease inG_(sα) localization to the processes and process tips between controlcells and desipramine-treated cells as 12% of the total cellular G_(sα)was located in the process tips of control cells versus 4% present inthe tips after desipramine treatment. TABLE 4 Percentage of G_(sα)Immunofluorescence Distribution in the Cell Sample Control Cell BodyProcesses C1 83.9 16.1 C2 89.0 11.0 C3 83.5 16.5 C4 95.6 4.4 Average88.0 ± 5.7 12.0 ± 5.7 Desipramine D1 91.4 8.6 D2 92.2 7.8 D3 100.0 0.0D4 100.0 0.0 Average 95.9 ± 4.7 4.1 ± 4.7

EXAMPLE 11 Antidepressant Induced G Protein α Subunit CellularRelocalization is Specific to G_(sα,)

To determine whether antidepressant-induced mobility is specific toG_(sα), G_(oα) distribution was examined in approximately 500 cellsunder the same treatment conditions. FIG. 9 shows that there was littleif any change in the distribution of G_(oα) after desipramine treatment.G_(oα) is throughout the cell without specific regions displaying anincreased staining intensity in control or treated cells. Some of thecontrol cells (FIGS. 9A and 9B) have a slight increase in stainingintensity at the process tips, but this is also seen in the treatedcells (FIGS. 9C and 9D), indicating that antidepressant treatment doesnot effect G_(oα) localization within the cell.

EXAMPLE 12 Fluoxetine Treatment also Promotes G_(sα) Migration

If the redistribution of G_(sα) is truly an antidepressant effect, thenother classes of antidepressant drug should have a similar effect.Confocal microscopic images of C6-2B cells treated with 10 μM fluoxetinefor three days show a similar Gsμ staining pattern compared withdesipramine-treated cells (FIG. 6A). The most striking similarity ofdesipramine and fluoxetine effects on G_(sα) localization is the loss ofstaining in the processes and process tips (compare FIGS. 6C and 6D, andFIG. 10A with FIGS. 6A and 6B). Approximately 100 cells were examinedfor qualitative differences as described above for FIG. 8. Of thefluoxetine-treated cells, 45% displayed intense staining in the processtips compared with the 64% of control and 32% of desipramine-treatedcells mentioned previously.

Chlorpromazine

The antipsychotic drug chlorpromazine was used as a control forantidepressant effects. When cells were treated with 10 μMchlorpromazine for three days, G_(sα) staining was evident throughoutthe cell body (FIG. 10B): there is G_(sα) immunostaining throughout thecell body, cell process, and process tip. This pattern of G_(sα)distribution was similar to other control cells; 68% of approximately100 cells demonstrated distinct staining in the cell processes andprocess tips.

Other Treatment Paradigms have a Similar Effect on G_(sα)

A lower dosage and longer exposure time for desipramine treatment (3 μMfor five day) was also tested. Control cells have intense staining atthe process tips, whereas the desipramine treated cells do not. The maindifference between the high-dose/three-day and the low-dose/five-daytreatment regimens is the cell body localization of G_(sα). A majorityof C6-2B cells treated with 10 μM desipramine display intense clusteringof GSA in the perinuclear region whereas cells treated with 3 μMdesipramine show a more even distribution between intense cell bodystaining and a more nondescript staining. One-day/10 μM desipraminetreatment of C6-2B cells resulted in a G_(sα) distribution similar tocells treated with 3 μM for five days (data not shown). Under the acutetreatment condition (one day, 10 μM) the number of cells lacking G_(sα)in the process tips was not significantly different from the controlcell population seen in Table 4 and FIG. 8

EXAMPLE 13 Screening for Effectiveness of Antidepressant Therapy andScreening for Agents Having Antidepressant Activity

In view of the foregoing results with respect to modifications noted inthe association of G_(sα) with components of the plasma membrane orcytoskeleton from glioma cells, this example is directed to a method fordetecting the effectiveness of antidepressant therapy as well methodsfor screening for agents having antidepressant activity.

An individual, diagnosed with major depression and receivingantidepressant therapy, may be assessed for the effectiveness of suchtherapy by the following method. Cells, for example, but not limited toblood cells [erythrocytes (red cells), leukocytes (white cells),platelets] and skin fibroblasts from peripheral tissues of the depressedindividual are collected and a determination is made as to whether therehas been a modification of the association of G_(sα) with components ofthe plasma membrane or cytoskeleton of cells from peripheral tissues ofthe depressed individual. Such modifications may include, but are notlimited to enhanced coupling between G_(sα) and adenylyl cyclase,redistribution of G_(sα) from a strongly hydrophobic region of theplasma membrane to a less hydrophobic membrane domain, and/orredistribution of G_(sα) from cell processes and process tips to thecell body.

Antidepressent therapy often requires about one month to begin toachieve effectiveness. Often multiple drugs must be employed before asatisfactory combination is stumbled upon. Any difference in suchmodifications (as compared to a normal or control state), when noted inthe early stages of antidepressant therapy and correlated with asubsequent decrease in the clinical depressive state would serve toquickly predict the success and/or failure of antidepressant therapy.More specifically, unlike psychological tests, if antidepressant therapywere to be effective, it is likely to increase such modifications in theassociation of G_(s)α with components of the plasma membrane orcytoskeleton within 3-5 days.

Further, the present invention is also useful for determining theeffectiveness of a putative antidepressant agent or agents, in that suchcompounds may be rapidly screened using the methods described herein.Specifically, putative agents are introduced in a cell culture whereinthe cells (for example, but not limited to Neuro2A cells(neuroblastoma), SKNSH cells (human blastoma) HEK293 cells (humanembroynic kidney cells after transfection with Type VI adenylyl cyclase)express Type VI adenylyl cyclase and a determination is made as towhether there has been a modification (for example, but not limited toenhanced coupling between G_(sα) and adenylyl cyclase, redistribution ofG_(sα) from a strongly hydrophobic region of the plasma membrane to aless hydrophobic membrane domain, and redistribution of G_(sα) from cellprocesses and process tips to the cell body of the association of G_(sα)with components of the plasma membrane or cytoskeleton of cells. Thoseagents that would be expected to have antidepressant activity would bethose compounds that increase the modifications in the association ofG_(sα) with components of the plasma membrane or cytoskeleton.

Further, the foregoing process lends itself to the use of fluorescenceresonance energy transfer (FRET) techniques for use as a high throughputsystems. We have recently developed a fluorescent analog of G_(sα) (aGPP fusion protein) for use in our experiments. Such an analog is usedwith a fluorescent adenylyl cyclase to determine the effects anantidepressant on the modification of interaction between Gsα andadenylyl cyclase. Specifically and in view of the foregoing examples anddiscussion, if the agent in question possessed antidepressant activityone would see an increase in FRET (an increased interaction between thefluorescent analog of G_(sα) and fluorescent adenylyl cyclase).

Although the present invention has been described in terms of preferredembodiments, it is intended that the present invention encompass-allmodifications and variations that occur to those skilled in the art uponconsideration of the disclosure herein, an in particular thoseembodiments that are within the broadest proper interpretation of theclaims and their requirements.

All literature cited herein is incorporated by reference.

1-40. (canceled)
 41. A method for assaying for an agent or agents havingan activity that modifies the association of Gsα with components of theplasma membrane or cytoskeleton of cells comprising the step of: (a)contacting said agent or agents with cultured cells; (b) determiningwhether there has been a modification of the association of Gsα withcomponents of the plasma membrane or cytoskeleton of the cells in step(a) via comparison to a control cell culture lacking said agent oragents; (c) identifying agents having antidepressant activity from adifference in the modification of the association of Gsα with componentsof the plasma membrane or cytoskeleton of the cells in step (a), whereinan agent or agents having antidepressant activity increases themodification of the association of Gsα with components of the plasmamembrane or cytoskeleton of the cells in step (a).
 42. A method forassaying for an agent or agents having antidepressant activitycomprising the step of: (a) contacting said agent or agents withcultured cells that expresses; (b) determining whether there has been amodification of the association of a fluorescent Gsα analog withcomponents of the plasma membrane or cytoskeleton of the cells in step(a) via comparison to a control cell culture lacking said agent oragents; (c) identifying agents having antidepressant activity from adifference in the modification of the association of the fluorescent Gsαanalog with components of the plasma membrane or cytoskeleton of thecells in step (a), wherein an agent or agents having antidepressantactivity increases the modification of the association of fluorescentGsα analog with components of the plasma membrane or cytoskeleton of thecells in step (a).
 43. The method of claim 42 wherein the modificationis enhanced coupling between the fluorescent Gsα analog and adenylylcyclase.
 44. The method of claim 43 wherein the adenylyl cyclase isfluorescently labeled.
 45. The method of claim 44, wherein theinteraction between the fluorescent Gsα analog and the fluorescentlylabeled adenylyl cyclase is monitored using fluorescence resonanceenergy transfer (FRET) wherein an increase in FRET is observed when saidagent or agents possess antidepressant activity.